In drug development, attrition rates are high with only one in five compounds making it through development to Food and Drug Administration approval (FDA) (DiMasi, J A, et al, J Health Econ 22,151-185, 2003). Moreover, despite dramatically increased investment, the rate of introduction of novel drugs has remained relatively constant over the past 30 years, with only two to three advances in new drug classes per year eventually making it to market (Lindsay M A, Nature Rev Drug Discovery, 2, 831-838, 2003).
Molecular and functional imaging applied to the initial stages of drug development can provide evidence of biological activity and confirm the putative drug having an effect on its intended target. Thus, there is considerable expectation that investment in molecular imaging technology will enhance drug development (Rudin M, Progress in Drug Res vol 62). The advantage of molecular imaging techniques over more conventional readouts is that they can be performed in the intact organism with sufficient spatial and temporal resolution for studying biological processes in vivo. Furthermore, it allows a repetitive, non-invasive, uniform and relatively automated study of the same biological model at different time points, thus increasing the statistical power of longitudinal studies plus reducing the number of animals required and thereby reducing cost of drug development.
Molecular Imaging
Molecular imaging refers to the convergence of approaches from various disciplines (cell and molecular biology, chemistry, medicine, pharmacology, physics, bioinformatics and engineering) to exploit and integrate imaging techniques in the evaluation of specific molecular processes at the cellular and sub-cellular levels in living organism. (Massoud T. F., Genes Dev. 17:545-580, 2003)
The advent of genetic engineering has brought about major changes to applied science, including for example the drug discovery pipeline. In the same way, the development and exploitation of animal imaging procedures is providing new means for pre-clinical studies (Maggie A. and Ciana P., Nat. Rev. Drug Discov. 4, 249-255, 2005). Animal models traditionally have been cumbersome because of the difficulty in quantifying physiological events in real-time. Over the years new imaging methods have been developed to overcome this difficulty, such as magnetic resonance imaging (MRI) and positron emission tomography (PET). More recently bioluminescence imaging based on in vivo expression of luciferase, the light-emitting enzyme of the firefly, has been used for non-invasive detection.
Molecular Imaging: Bioluminescence
In vivo bioluminescent imaging (BLI) is a sensitive tool that is based on detection of light emission from cells or tissues. The utility of reporter gene technology makes it possible to analyze specific cellular and biological processes in a living animal through in vivo imaging methods. Bioluminescence, the enzymatic generation of visible light by a living organism, is a naturally occurring phenomenon in many non-mammalian species (Contag, C. H., et al, Mol. Microbiol. 18:593-603, 1995). Luciferases are enzymes that catalyze the oxidation of a substrate to release photons of light (Greer L. F., Ill, Luminescence 17:43-74, 2002). Bioluminescence from the North American firefly is the most widely studied. The firefly luciferase gene (luc) expression produces the enzyme luciferase which converts the substrate D-luciferin to non-reactive oxyluciferin, resulting in green light emission at 562 nm. Because mammalian tissues do not naturally emit bioluminescence, in vivo BLI has considerable appeal because images can be generated with very little background signal.
BLI requires genetic engineering of cells or tissues with an expression cassette consisting of the bioluminescent reporter gene under the control of a selected gene promoter constitutively driving the light reporter (FIG. 3). In order to induce light production, the substrates such as luciferin are administered by intracerebroventricular (icv), intravenous (iv), intraperitoneal (ip) or subcutaneous (sq) injection.
The light emitted by luciferase is able to penetrate tissue depths of several millimeters to centimeters; however photon intensity decreases 10 fold for each centimeter of tissue depth (Contag, C. H., et al, Mol. Microbiol. 18:593-603, 1995). Sensitive light-detecting instruments must be used to detect bioluminescence in vivo. The detectors measure the number of photons emitted per unit area. Low levels of light at wavelengths between 400 and 1000 nm can be detected with charge coupled device cameras that convert the light photons that strike silicon wafers into electrons (Spibey C P et al electrophoresis 22:829-836, 2001). The software is able to convert electron signals into a two-dimensional image. The software is also able to quantify the intensity of the emitted light (number of emitted photons striking the detectors) and convert these numerical values into a pseudocolor graphic or grayscale image (FIGS. 2A and 2B). The actual data is measured in photons, but the pseudocolor graphic enables rapid visual interpretation. Quantitative measurements within a region of interest may be necessary for more subtle differences. The use of cooled charge coupled device (CCD) cameras reduces the thermal noise and a light-tight box allows luciferase-produced light to be optimally visualized and quantified (Contag C. H. and Bachmann, M. H., Annu. Rev. Biomed. Eng. 4:235-260, 2002). It is useful to have the luciferase image superimposed on another type of image such as an autograph or radiograph for anatomical location of the emission signal (FIG. 2B). The software superimposes images for visualization and interpretation.
By combining animal engineering with molecular imaging techniques, it has become possible to conduct dynamic studies on specific molecular processes in living animals. This approach could potentially impact on pre-clinical protocols thus widely changing all aspects of medicine (Maggie A. Trends Pharmacolo. Sci 25, 337-342, 2004)
G-Protein Coupled Receptors (GPCRs)—GPCRs as Drug Targets
GPCRs constitute a large super family of cell surface receptors that are classified into more than 100 subfamilies on the basis of their shared topological structure; GPCRs are also referred to as seven transmembrane (7TM) receptors. GPCRs are the most frequently addressed drug targets in the pharmaceutical industry. Approximately 30% of all marketed prescription drugs target GPCRs, which makes this protein family pharmaceutically the most successful target class (Jacoby, E; Chem. Med. Chem., 1: 761-782, 2006).
The interaction between GPCRs and their extracellular ligands has proven to be an attractive point of interference for therapeutic agents. For this reason, the pharmaceutical industry has developed biochemical drug discovery assays to investigate these ligand-GPCR interactions. Interaction of an activated GPCR with a heterotrimeric G-protein catalyzes the exchange of guanosine diphosphate (GDP) by guanosine triphosphate (GTP) enabling the interaction with several downstream effectors (Cabrera-Vera T. M., Endocr. Rev. 24:765-781, 2003). Signaling downstream is dependent on the G-alpha isoform that is preferred by the GPCR of interest. Proteins of the G-alphaq/11 family stimulate phospholipase C (PLC), while representatives of the G-alphai/0 and G-alphas families mostly modulate adenylate cyclase (AC) activity. If the GPCR of interest signals via PLC, then the most broadly applied reporter based technique to measure GPCR activation is a calcium (Ca+2) release assay, either measured in a fluorescent format using Ca+2-sensitive fluorophores (Sullivan E, Methods Mol. Biol. 114:125-133, 1999) or in a luminescent format using aequorin and a chemiluminescent substrate (Dupriez V. J., Receptors Channels 8: 319-330, 2002). If the GPCR of interest signals via AC, then cytosolic cyclic adenosine monophosphate (cAMP) content may be determined using various detection technologies (Gabriel D. Assay Drug Dev. Technol. 1:291-303, 2003)
GPCR reporter based assays have been extensively used in current drug discovery programs. Typically, GPCR reporters have been introduced into cell based systems to support in vitro high-throughput screening (HTS) of large pharmaceutical libraries to identify ligands or compounds that activate or module the specific GPCR. Secondary and follow-up cell based assays confirm and refine any “hits” identified in HTS against a specific GPCR; but again, these assays rely on recombinant DNA methods to introduce a cloned GPCR into a transformed cell type. While transformed cell types have excellent proliferative capacity to support large screening programs, they often display aberrant genetic and functional characteristics and consequently significant attrition of putative “hits” from HTS is encountered using this paradigm.
For several years, bioluminescence-based reporter gene assays have been employed to measure functional activity of GPCRs (Hill, S. J. Curr. Opin. Pharmacol 1: 526-532, 2001). This assay format is very sensitive owing to the low signal background of the bioluminescent readout and the signal amplifications steps between GPCR activation and the cumulative reporter gene expression.
A cAMP response element (CRE) in the promoter of the reporter gene enables the specific monitoring of G protein dependent signaling. When a ligand binds to the GPCR it causes a conformational change in the GPCR which allows it to activate an associated G-protein. The enzyme adenylate cyclase is a cellular protein that can be regulated by G-proteins. Adenylate cyclase activity is either activated or inhibited when it binds to a subunit of the activated G protein. Signal transduction depends on the type of G protein. Adenylate cyclase acts to either increase or decrease cAMP production in the cell. The cAMP produced is a second messenger in cellular metabolism and is an allosteric activator to protein kinase A (PKA). When there is no cAMP, the PKA complex is inactive. When cAMP binds to the regulatory subunits of PKA, their conformation is altered, causing the dissociation of the regulatory subunits, which activates protein kinase A and allows further biological effects. PKA then phosphorylates and activates the transcription factor CREB. CREB binds to certain DNA sequences called cAMP response elements (CRE) and thereby increases or decreases transcription, and thus the expression, of certain genes, such as the luciferase reporter gene.
The CreLuc transgene is designed to assay activation of all three major GPCRs either directly through the cAMP intracellular signaling pathway or indirectly through signaling via PLC. Because any one cell type contains many different types of GPCRs on their cell surface, (thus any cell would have GPCRs signaling via G-alphaq/11, G-alphai/0 and G-alphas occurring simultaneously within a cell) conventional wisdom would suggest that it would be improbable that a transgene such as CreLuc would be specific enough to discriminate any one specific GPCR ligand. However, we demonstrate here the CreLuc transgene is able to discriminate GPCR ligands. We predict that the bioluminescent signal for the luciferase reporter in cells, tissues slices, or the whole animal will be increased with forskolin and be modulated by ligands for Gs, Gq or Gi receptors. Table 1 shows the anticipated effect that GPCR activation/inhibition will have on the CreLuc reporter system upon binding to a GPCR ligand. Further, we show data that our novel CreLuc reporter system can discriminate different classes of GPCR ligands and that such a reporter system is applicable for identifying novel GPCR ligands when used in cells, tissue slices and the whole animal.
TABLE 1Predicted change in bioluminescent signal from the CreLuc reporterupon ligand binding to specific GPCRsReceptor TypeAgonistAntagonist; Inverse AgonistGs; GqIncreaseDecreaseGiDecreaseIncreaseA GPCR Bioimaging Reporter Transgenic Model
Significant attrition of potential drug candidates in the current drug discovery paradigm is encountered in the phase transition from cell-based reporter assays to in vivo models. Numerous in vivo models are available that recapitulate either all or part of a particular human disease. Demonstrating lead compound activity in these models is a significant milestone for progression of new chemical GPCR drugs. Animal disease models typically require a large number of animals and time to allow for the development of their phenotype and an accurate assay of the candidate compound's impact on altering the disease outcome. Following in vitro testing, the next level of testing a drug candidate in a complex system is using in vivo testing or in vivo models of disease states which are mechanistic based. Failures to alter the induced disease outcomes are poorly understood but yet result in the large attrition rates of candidate compounds in the drug development pipeline.
A transgenic model containing a GPCR ligand binding and activation reporter assay would be a significant improvement in the current drug discovery paradigm for GPCRs. For instance, an embodiment of this invention describes a transgene containing the cAMP reporter assay based on a luciferase reporter (CreLuc) that is combined with molecular imaging in whole animals, tissues, or cells which would significantly accelerate GPCR ligand drug discovery (Bhaumik, S. and Gambhir, S. S., Proc. Natl. Acad. Sci. USA, 99:377-382 2002; Hasan M. T., et al., Genesis 29:116-122, 2001). As described herein, embodiments of the transgenic non-human animal of the instant invention offer the following non-limiting advantages:                1. Tissues or cell based assays have the same reporter system as in the transgenic in vivo model assay thus reducing the number of unknowns in complex intact biological systems.        2. Non-invasive imaging allows quantitative analysis of ligand or compound activity in a time-course assay in the same animal.        3. Non-invasive imaging reduces the number of animals per study and leads to greater statistical power by each animal being its own control wherein the control would be the animal assayed at time zero.        4. The transgenic animal would be a source of cells and tissues to support parallel assays done in vitro or ex vivo.        5. The assay of the transgenic animal would support the assay of ligand activity in native cell types which leads to a more realistic profile of ligand: receptor interaction.        6. The transgenic animal allows for simultaneous assessment of pharmacodynamics and pharmacokinetics of GPCR ligands.        7. The transgenic animal allows for simultaneously identification of tissue and cell-type specificity at either the organ or whole animal level.        8. The transgenic animal allows for cross breeding with other genetically altered models to reveal novel signaling pathways and their response to specific ligands.        
Many transgenic animals engineered with different reporters are being employed in the study of molecular processes such as drug metabolism (Zhang W., et al. Drug Metab. Dispos. 31:1054-1064, 2003), genotoxicity (Gossen J. A., et al., Proc. Natl. Acad. Sci. USA 86:7971-7975, 1989) and the effects of toxic compounds (Sacco M. G. et al., Nat. Biotechnol 15:1392-1397, 1997). To achieve their design goals, a GPCR reporter animal suitable for molecular imagining studies has to incorporate several elements arranged to allow both high levels of reporter expression to support a large window of bioluminescent detection as well as expression in every cell type to support broad acute in vivo assays on biodistribution of the ligand or compound under study.
The complexity and diversity of the mechanisms involved in gene expression will never allow researchers to construct genes capable in all cases of being expressed in transgenic animals in a fully predictable manner (Pinkert, C. A. (ed.) 1994. Transgenic animal technology: A laboratory handbook. Academic Press, Inc., San Diedo, Calif.; Monastersky G. M. and Robl, J. M. (ed.) (1995) Strategies in Transgenic Animal Science. ASM Press. Washington D.C). Only through extensive trial and error can unique combinations of transgene structures be arrived at to deliver model design goals as required for bioimaging of GPCR reporters.
Utility of a Transgenic GPCR Reporter over Recombinant Cell Assays
As screening technology advances to the point of understanding the behaviors of individual GPCRs, it is clear that rather than being on-off switches, these receptors are acting more as microprocessors of information. This has introduced the phenomenon of functional selectivity, whereby certain ligands initiate only portions of the signaling mechanism mediated by a given receptor, which has opened new horizons for drug discovery. The need to discover new GPCR ligand relationships and quantify the effect of the drug on these complex systems to guide medicinal chemistry puts significantly higher demands on any pharmacological reporter assay. This concept drives the return to whole-system assays from the reductionist recombinant cell based screening systems. Profiling a ligand's activity with a specific GPCR or set of GPCRs in a native cellular environment is expected to improve the success rate of identifying new drugs against a key class of pharmaceutically important receptors (Kenakin T P, Nat. Rev. Drug Discov. 8,617-625, 2009) An animal model containing a bioluminescent GPCR reporter transgene is a highly desirable molecular imaging strategy to define GPCR ligand activity in an intact biologically complex system with the goal of improving drug discovery to fight human diseases.
Because activation of CRE/CREB is involved many varied biological processes, there has been considerable interest in studying the activation of CRE by using a CRE/CREB reporter expression system. Cyclic adenosine monophosphate (cAMP) is a second messenger in intracellular signal transduction following receptor activation and subsequent activation of protein kinase, thereby being involved in the regulation of many biological processes. CREB (cAMP responsive element binding protein), phosphorylated by kinase activated by cAMP, binds to the cAMP responsive element (CRE) in the promoter region of many genes and activates transcription (Shaywitz and Greenberg, Annul. Rev. Biochem., 68:821-861, 1999). Transgenic mice carrying six tandem CREs with a minimal herpes simplex virus (HSV) promoter driving beta-galactosidase expression were used to study CRE-mediated gene expression in brain slices in response to chronic antidepressant treatment (Thome J., et al., J. Neurosci. 20:4030-4036, 2000). Similarly, transgenic mice carrying four copies of rat somatostatin gene promoter CRE fused to a thymidine kinase promoter and the luciferase gene have been used to study CRE activation in histological brain slices or homogenates (Boer et al, PloS One, May 9; 2(5):e431, 2007). However, studies to date have been hampered by the need to screen large numbers of transgenic lines to find a suitable animal model. Further, after the appropriate line has been identified, relatively low reporter expression levels require the transgenic animal be euthanized in order to measure the reporter gene, thus requiring large number of animals be used to in a single experimental paradigm.
An embodiment of the invention is the development of a transgene comprising insulator elements, response elements, promoter elements, reporter genes, and functional elements. The transgene can be quickly introduced into non-human animals because of its high rate of integration and high level of reporter gene expression, thus transgenic animals can be easily developed as models to study regulatory element activation in vivo (i.e., in the living animal), in situ (e.g., brain slices, intact whole organ) or in vitro (e.g., primary cells cultured from the transgenic animal, tissue homogenates).
An embodiment of the invention is a transgene comprising a CRE Luc reporter system used in transgenic non-human animals as models to quantify GPCR ligand activities through the regulation of intracellular cAMP levels in vivo. As a non-limiting example, we have demonstrated changes in the luciferase reporter via bioluminescence in isolated primary cells and in whole animals using general cAMP regulators. In another embodiment, activation of the reporter has been assayed and confirmed in tissue extracts using luciferase assays ex vivo. The response of the CRE Luc transgene has been documented in multiple mouse lines and exhibits either single or multiple tissue activation profiles. Furthermore, as non-limiting examples, we demonstrate that specific GPCR ligands activated the CRE Luc transgene in whole animals, tissue slices, and primary cells.